The previous cycle of this grant hypothesized a critical role for the inflammatory IKKbeta/NF-kappaB pathway in the pathogenesis of obesity- and diet-induced insulin resistance. We now know that the pathway is activated in fat and liver by obesity and high fat diet (HFD) and that activation of NF-kappaB in fat and liver causes insulin resistance, at least in part due to the production of proinflammatory cytokines (IL-6, resistin, IL-1beta, TNF-alpha). Inhibition of IKKbeta and NF-kappaB, either genetically or pharmacologically, and cytokine neutralization reverse insulin resistance in animals and/or humans. Elevations in inflammatory markers that are seen in patients are readily reproduced in rodent models dietary and genetically induced insulin resistance. These are reversed in both rodents and humans in parallel with improvements in insulin resistance and dramatic reductions in triglyceride, free fatty acid and glucose levels. To continue developing and testing these hypotheses, we now propose a comprehensive plan to identify intracellular proteins that modulate NF-kappaB signaling and insulin resistance. In addition to the cytokines listed above, other NF-kappaB targets induced by HFD and obesity in fat and liver are associated with the metabolic syndrome and atherosclerosis (CRP, PAI-1, SAA-1, VCAM1, ICAM1, iNOS and COX2). As the subject of this proposal we also see constitutive, NF-kappaB dependent expression of A20, IKKi/epsilon, and IkappaBzeta, in fat and liver of obese/HFD rodent models. These normally inducible regulators and/or mediators of NF-kappaB signaling have intriguing functions in host defense. We plan to determine if they also function in insulin resistance. A20 induction limits NF-kappaB signaling by altering the ubiquitination of upstream signaling proteins; NF-kappaB induces the expression of IKKi/epsilon, a Ser/Thr kinase that inhibits insulin signaling; and IkappaBzeta, unlike IkappaBalpha, selectively increases expression of a few interesting NF-kappaB targets such as IL-6. Experiments presented in this application determine the in vivo roles of these proteins in fat and liver, delving further into the mechanisms of HFD/obesity-induced insulin resistance by looking both at tissue-specific effects and inter-organ cross-talk. The findings will improve our understanding of the role of subacute 'inflammation' in insulin resistance, T2D and the metabolic syndrome, and may identify new and more selective targets for therapeutic intervention.